Composite For Liver-Specific Delivery and Release of Therapeutic Nucleic Acids or Drugs

ABSTRACT

The inventive composite having a nanoscale particle size can specifically deliver therapeutic nucleic acids or drugs to the liver and selectively release them into hepatic cells to manifest potent therapeutic effects without inducing any enzymatic abnormalities or pathological damage to the normal liver function, when administered together with the therapeutic agents.

FIELD OF THE INVENTION

The present invention relates to a composite for liver-specific deliveryof a therapeutic nucleic acid or a drug, a process for preparing thesame and a composition comprising the same with a pharmaceuticallyacceptable carrier.

BACKGROUND OF THE INVENTION

A tissue-specific gene and drug delivery system has long been consideredimportant for drug discovery and pharmaceutical advancement because mostdrugs are systemically delivered and circulated in the body whenadministered to a patient, which might adversely affect healthy organsor cells. The tissue-specific delivery system allows the accumulation ofa high drug concentration at the target tissue which eliminating adverseside effects, leading to efficient treatment of tissue-specificdiseases.

Some liver diseases arise from infection by pathogenic viruses, e.g.,HBV (hepatitis B virus) and HCV (hepatitis C virus), whilenon-infectious liver diseases result from exposure to liver-toxicmaterials, or genetic or environmental disorders. The progression ofearly-stage liver diseases caused by biological stimuli ultimately leadsto chronic hepatitis, liver cirrhosis or hepatocellular carcinoma (HCC).Among several drug or gene delivery systems currently studied in thetreatment of such liver diseases, a lipoprotein system, mainly that ofHDL (high density lipoprotein), has advantages over other deliverysystems which use viral vectors (Wang X., et al., Gene Ther. (2006), 13:1097-1103), non-viral complexes (Landen C. N., et al., Cancer Res.(2005), 65: 6910-6918; Morrissey D. V., et al., Nat. Biotechnol. (2005),23: 1002-1007; Sorensen D. R., et al., J. Mol. Biol. (2003), 327:761-766; and Urban-Klein B., et al., Gene Ther. (2005), 12: 461-466) andantibodies (Song E., et al., Nat. Biotechnol. (2005), 23: 709-717). Forexample, the lipoprotein can be preferably recognized and taken up viacell surface receptors specific for liver cells (Firestone R. A.,Bioconjug. Chem. (1994), 5: 105-113; de Smidt P. C., et al., Crit. Rev.Ther. Drug Carrier Syst. (1990), 7: 99-120; and Filipowska D., et al.,Cancer Chemother Pharmacol. (1992), 29: 396-400), and it is anendogenous product which is not detrimental to human and does nottrigger immunological responses in clinical applications (Pussinen P.J., et al., Biochem. Biophys. Acta. (2000), 1485: 129-144).

Recently, there has been a report that a recombinant high densitylipoprotein (HDL) can be used as a carrier for delivering a lipophilicantitumor drug into human hepatocellular carcinoma cells by takingadvantage of the hydrophobic cholesterol ester-loading properties of HDL(Lou B., et al., World J. Gastroenterol. (2005), 11: 954-959). However,it has merely been demonstrated in vitro, but not in vivo, that thecellular uptake of the HDL carrier by a hepatoma cell line, SMMC-7721,is higher in compared with a normal liver cell line, L02, and thelimitation in tissue-specific targeting remains to be solved.

The present inventors have therefore endeavored to develop an effectivesystem for liver-specific delivery of a therapeutic drug, and have foundthat a composite comprising an apolipoprotein A-I and a liposome-formingmaterial can specifically deliver and release therapeutic drugs to theliver.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide acomposite capable of specifically delivering and releasing a therapeuticnucleic acid or a drug to the liver when administered via a systemicroute.

It is another object of the present invention to provide a process forthe preparation of said system.

It is further object of the present invention to provide a compositionfor liver-specific delivery of a therapeutic nucleic acid or drug,comprising said composite.

In accordance with one aspect of the present invention, there isprovided a composite comprising an apolipoprotein A-I (Apo A-I) and aliposome-forming material.

In accordance with another aspect of the present invention, there isprovided a process for the preparation of the composite.

In accordance with further another aspect of the present invention,there is provided a composition comprising the composite and apharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects and features of the present invention willbecome apparent from the following description of the invention, whentaken in conjunction with the accompanying drawings, which respectivelyshow:

FIG. 1A: Purified human Apo A-I from adult blood separated by 4-20%sodium dodecyl sulfate-polyacrylamide gel electrophoresis;

FIG. 1B: In vivo images of a mouse intravenously injected with Apo A-Ilabeled with an infrared fluorescent dye at several times after theinjection;

FIG. 1C: Percent uptake rate of Apo A-I in the livers of mice injectedwith Apo A-I at different time points (n=4) after the injection;

FIG. 2A: Whole body images for radioiodine signals captured using agamma camera in a mouse intravenously injected with the inventivecomposite (DTC-Apo*/RLuc) which contains a Renilla luciferase expressionplasmid, phRL-CMV and ¹³¹I label;

FIG. 2B: Whole body images captured several times after mice wereintravenously injected with the inventive composite (DTC*-Apo/RLuc) anda comparative composite (DTC*/RLuc), which are labeled with rhodamine,respectively;

FIG. 2C: Luciferase levels measured in tissue homogenates from heart,lung, kidney and liver of mice (n=3) which were intravenouslyadministered with a mock control (5% dextrose), naked DNA, or theinventive or a comparative composite containing a Renilla luciferaseexpression plasmid, phRL-CMV (DTC/RLuc or DTC-Apo/RLuc);

FIG. 3A: Relative levels of secreted HBsAg determined by ELISA in micewhich were intravenously injected with a mock control (5% dextrose);naked siHBV; comparative composites containing HBV-specific siRNA(DTC/siHBV) and irrelevant control siRNA (DTC/siCont); and the inventivecomposites containing HBV-specific siRNA (DTC-Apo/siHBV) and irrelevantcontrol siRNA (DTC-Apo/siCont), respectively, at days 2, 4, 6 and 8after the injection;

FIG. 3B: Serum HBsAg levels measured by ELISA in in vivo mouse models ofHBV replication which were intravenously injected with the inventivecomposites containing HBV-specific siRNA (DTC-Apo/siHBV) and irrelevantcontrol siRNA (DTC-Apo/siCont), respectively, at doses of 0.5, 1 or 2mg/kg, at day 4 after the injection;

FIG. 4A: In vivo images of luciferase gene expression in mice which wereadministered with a luciferase expression plasmid, pEGFPLuc, and one dayafter administration, intravenously injected with the inventivecomposites containing luciferase-specific siRNA (DTC-Apo/siLuc) orirrelevant control siRNA (DTC-Apo/siCont), respectively; and

FIG. 4B: Relative luciferase expression levels measured by countingbioluminescent signals emitted from the liver of the mice shown in FIG.4A.

DETAILED DESCRIPTION OF THE INVENTION

The composite of the present invention may be in the form ofnanoparticles having an average particle size ranging from 50 to 400 nm,preferably 100 to 250 nm, and the apolipoprotein A-I (Apo A-I) used inthe present invention may be obtained from human blood by cold ethanolprecipitation in accordance with a conventional method (e.g., Lerch, P.G., et al., Vox. Sang. (1996), 71: 155-164).

The liposome-forming material employed in the inventive composite may bea cationic or neutral liposome-forming material, or a mixture thereof,which play a role of avoiding undesirable interactions between theinventive composite and unknown serum components. Representativeexamples of the cationic liposome-forming material include DOTAP(1,2-dioleoyl-3-trimethylammonium-propane), DC-cholosterol(3β-[N-(N′,N′-dimethylaminoethane)-carbamyl]cholesterol), DDAB(dimethyldioctadecylammonium bromide), and a mixture thereof, and theneutral liposome-forming material may be DOPE (L-alpha-dioleoylphosphatidylethanolamine), cholesterol, or a mixture thereof.

The inventive composite may comprise Apo A-I and the liposome-formingmaterial at a weight ratio ranging from 1:0.1 to 1:1000, preferably 1:1to 1:100.

The composite of the present invention may further comprise atherapeutic nucleic acid and/or drug.

The therapeutic nucleic acid may be a DNA such as plasmid and PCRproduct, an RNA such as siRNA and ribozyme, or a derivative thereofobtained by chemical modification, preferably siRNA specific for HBV orHCV genome.

The therapeutic drug may be an active polypeptide, anticancer agent, orantivirus agent, which does not limit the scope of the presentinvention.

The active polypeptide used in the inventive composition may be selectedfrom the group consisting of epidermal growth factor (EGF),erythropoietin (EPO), coagulation factors VIII, IX and VIIa, folliclestimulating hormone (FSH), granulocyte colony-stimulating factor (GCSF),granulocyte-macrophage colony stimulating factor (GM-CSF), insulin,insulin-like growth factor (IGF), interferon-α, -β and -γ (IFN-α, -β and-γ), interleukin-1, -2, -11, -12 and -15 (IL-1, -2, -11, -12 and -15),parathyroid hormone (PTH), platelet-derived growth factor (PDGF), humangrowth hormone (hGH), tissue plasminogen activator (tPA), vascularendothelial growth factor (VEGF), and a mixture thereof, which does notlimit the scope of the present invention.

Further, the anticancer agent may be selected from the group consistingof carboplatin, cisplatin, oxaliplatin, heptaplatin, etoposide,semustine, hydroxycarbamide, citarabine, fludarabine, doxorubicin,epirubicin, idarubicin, pirarubicin, fluorouracil (5-FU), fluoxuridine,mitomycin, bleomycin, clofazimine, interferon, streptozocin,gemcitabine, enocitabine, capecitabine, ursodeoxycholic acid, sorafenib,tegafur, holmium and a holmium-chitosan complex, and the antivirus agentmay be selected from the group consisting of atazanavir, ribavirin,zanamivir, acyclovir, entecavir, didanosin, nevirapine, valaciclovir,nelfinavir, efavirenz, ganciclovir, lamivudine, famciclovir, stavudine,abacavir, indinavir, oseltamivir, inosiplex, and adefovir, which doesnot limit the scope of the present invention.

The composite of the present invention may be prepared by a methodcomprising (i) dispersing liposome-forming materials in an organicsolvent to form a liposome, (ii) dispersing the liposome in a dextrosesolution, and sonicating the mixture to obtain a liposome suspension,and (iii) adding a solution containing Apo A-I thereto to allow formingthe inventive composite. The method of the present invention may furthercomprise (iv) adding a therapeutic nucleic acid or a drug to thesuspension of the inventive composite obtained in step (iii).

In accordance with further aspect of the present invention, there isprovided a composition for liver-specific delivery of a therapeuticnucleic acid or drug, comprising the inventive composite and apharmaceutically acceptable carrier. The inventive composition mayfurther comprise the therapeutic nucleic acid or drug as describedabove.

The composition of the present invention may be formulated for oral orparenteral administration according to any one of the procedures wellknown in the art, so as to take the form of sterilized aqueous solution,hydrophobic solvent, suspension, emulsion, lyophilized formulation orsuppository. In the formulation of the inventive composition, thehydrophobic solvent or suspension may further comprise a vegetable oilsuch as propylene glycol, polyethylene glycol and olive oil; an estersuch as ethyloleate; or a mixture thereof, and the suppository mayfurther comprise witepsol, macrogol, Tween 61, cacao butter, laurel oil,glycerol, gelatine, or a mixture thereof.

Further, a proposed daily dose of the composition of the presentinvention for administration to a human (of approximately 70 kg bodyweight) is about from 0.1 mg to 1000 mg, more preferably about from 1 mgto 500 mg. It should be understood that the daily dose should bedetermined in light of various relevant factors including the conditionto be treated, the severity of the patient's symptoms, the route ofadministration, or the physiological form of the anticancer agent; and,therefore, the dosage suggested above does not limit the scope of theinvention in anyway.

The following Examples are intended to further illustrate the presentinvention without limiting its scope.

TEST EXAMPLE 1 Liver-specificity of Purifed Apo A-I

High purity human apolipoprotein A-I (Apo A-I, 28 kDa) was obtained fromserum fractions of normal healthy adults not infected with viralpathogens such as HBV, HCV or HIV by cold ethanol precipitationaccording to the established protocol (Lerch, P. G., et al., Vox. Sang.(1996), 71: 155-164).

After sodium dodecyl sulfate-polycrylamide gel electrophoresis(SDS-PAGE), and the purified Apo A-I was characterized by Coomassie bluestaining. The result is shown in FIG. 1A. The identity of the purifiedApo A-I was confirmed by western blot analysis using a goat anti-humanApo A-I antibody (Academy Biomedical Company, USA) which hascross-reactivity to mouse Apo A-I, and a secondary antibody, rabbitanti-goat IgG-HRP (KPL, USA).

For in vivo imaging, the purified protein (0.6 mg) was labeled with aninfrared dye using IRDye 800 CW in vivo imaging agent (LI-CORBiosciences, USA), and purified using a dextran desalting column (PierceBiotechnology, Inc., USA) to remove unincorporated dye. The labeled ApoA-I (200 μg) was administered to 6- to 8-week-old female nude mice(Charles River Laboratories, Inc., USA) via tail vein injection. After7, 40, 90, 150, 240 or 360 min, the test mice anesthetized with 2%isoflurane were placed in a supine position in a light tight chamber,and their whole body images were obtained using IVIS 200 imaging system(Xenogen, USA) and Living Image Software (Xenogen, USA). The resultingimages are shown in FIG. 1B.

As shown in FIG. 1B, Apo A-I can be specifically delivered to and stablymaintained in the liver for at least 6 hours when systemicallyadministered.

Further, the photon intensities in the liver of the test mice weremeasured using Living Image Software (Xenogen, USA) at each time pointafter the administration, and the result is shown in FIG. 1C.

FIG. 1C reveals that the uptake yield of the administered Apo A-I byliver are maximal at approximately 45% within 150 min afteradministration.

Taken together, these data demonstrate that the purified Apo A-Imaintains its native conformation required for cell-surface receptorrecognition and catabolic circulation in vivo, suggesting that it mightbe applicable as a potent candidate carrier for targeting the liver infeasibility studies for therapeutic drug delivery.

EXAMPLE 1 Preparation of the Inventive Composite

An equimolar mixture of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP;Avanti Polar Lipids, USA) and cholesterol (Sigma, USA) was dispersed inchloroform and mixed to form a cationic liposome of DOTAP/cholesterol(DTC). After the liposome assembly was formed, the organic solvent wasremoved by evaporation under a stream of N₂ gas and the residue was keptin a vacuum desiccator for 2 hours to ensure the removal of the residualorganic solvent. The resulting dried film was hydrated in a 5% dextrosesolution and the suspension thus obtained was sonicated using a bathsonicator. A solution containing 10% the Apo A-I purified in TestExample 1 was added thereto at a DTC: Apo A-I mix ratio of 10:1 (w/w),and the mixture was kept overnight at 4° C. to obtain the inventivecomposite (DTC-Apo).

EXAMPLE 2 Preparation of the Inventive Composite Containing Nucleic Acid

40 μg of HBV X gene-specific siRNA (SEQ ID NOs: 1 (sense) and 2(antisense); Shin, D., Virus Res. (2006), 119: 146-153) was mixed with400 μg of the inventive composite, DTC-Apo, in 200 μl of 5% dextrosesolution and the mixture was incubated at room temperature for 30 min,to obtain the inventive composite containing HBV X gene-specific siRNA,named DTC-Apo/siHBV.

EXAMPLE 3 Preparation of the Inventive Composite Containing Nucleic Acid

The procedure of Example 2 was repeated except for using fireflyluciferase-specific siRNA (SEQ ID NOs: 3 (sense) and 4 (antisense);Elbashir, S. M., Nature (2001), 411: 494-498) instead of HBV-specificsiRNA, to obtain the inventive composite containing fireflyluciferase-specific siRNA, named DTC-Apo/siLuc.

EXAMPLE 4 Preparation of the Inventive Composite Containing Nucleic Acid

The procedure of Example 2 was repeated except for using 0.3 mg of aplasmid phRL-CMV encoding Renilla luciferase (Promega, WI) and 3 mg ofDTC-Apo instead of 40 μg of HBV-specific siRNA and 400 μg of DTC-Apo, toobtain the inventive composite containing a plasmid phRL-CMV, namedDTC-Apo/RLuc.

EXAMPLE 5 Preparation of the Inventive Composite Containing Nucleic Acid

The procedure of Example 2 was repeated except for using control doublestranded RNA (SEQ ID NOs: 5 (sense) and 6 (antisense)) instead ofHBV-specific siRNA, to obtain the inventive composite containing controldouble stranded RNA, named DTC-Apo/siCont.

COMPARATIVE EXAMPLES 1 TO 4 Preparation of the Comparative CompositeContaining Nucleic Acid

The procedures of Examples 2 to 5 were repeated except for using DTCinstead of DTC-Apo, to obtain comparative composites named DTC/siHBV,DTC/siLuc, DTC/RLuc and DTC/siCont, respectively.

TEST EXAMPLE 2 Characterization of the Inventive and ComparativeComposite

The inventive and comparative composites obtained in Examples 2 to 5 andComparative Examples 1 to 4 were characterized by measuring their sizeand charge using a Zetasizer 3000 apparatus (Malvern Instruments,Malvern, Worcestershire, United Kingdom), respectively, to determine theaverage diameters and zeta potential values thereof. The results areshown in Table 1.

TABLE 1 Formulation Size (nm) ζ pot (mV) DTC 176.5 ± 1.4 53.3 ± 4.0 DTCwith DNA 205.5 ± 4.2 42.7 ± 1.8 DTC with siRNA 196.0 ± 1.8 44.6 ± 2.2DTC-Apo 147.9 ± 2.8 49.5 ± 6.3 DTC-Apo with DNA 179.5 ± 3.4 38.6 ± 4.0DTC-Apo with siRNA 177.1 ± 1.4 39.1 ± 2.8

As shown in Table 1, the inventive composite has an average particlesize in the nanoscale range, suitable for systemic administration, andit is positive charged no matter whether it contained a nucleic acid ornot, which showed that the inventive composite would not occurundesirable interaction with unknown serum components.

TEST EXAMPLE 3 Liver-specific Gene Delivery In Vivo

In order to facilitate the systemic and sensitive detection of the invivo migration route, Apo A-I of DTC-Apo/RLuc obtained in Example 4 waslabeled with 0.6 mCi ¹³¹I (The Korea Atomic Energy Research Institute,Daejeon, South Korea) by the chloramines-T method (named DTC-Apo*/RLuc).200 μCi of the purified DTC-Apo*/RLuc was intravenously injected intonude mice (Charles River Laboratories), and the radioactivity from thewhole body of each mouse was monitored using a gamma-camera (Medicalimaging Electronics, USA) at 40, 120 and 240 min postinjection,respectively. The results are shown in FIG. 2A.

FIG. 2A clearly shows that the inventive composite is accumulated in thehepatic tissue at 40 min after administration.

Further, the cationic liposomes of the comparative and inventivecomposites, DTC/RLuc and DTC-Apo/RLuc obtained in Comparative Example 3and Example 4, respectively, were labeled with a fluorescent dye,rhodamine using lissamine rhodamine B-diacyl phosphatidylethanolamine(Avanti Polar Lipids), to obtain labeled composities, DTC*/RLuc andDTC*-Apo/RLuc, respectively. The labeled composites were each injectedinto nude mice (Charles River Laboratories), and the whole body wasmonitored using IVIS 200 imaging system (Xenogen, USA) at 20, 60 and 100min postinjection.

As shown in FIG. 2B, the accumulation level of the inventive compositeis enhanced in the liver more prominently than that of the comparativecomposite. The fluorescent signal noise detected at the ends of the limbmay be due to the overlapped emission wavelengths between rhodamine andred blood cells.

Further, in order to examine nucleic acid release by the inventivecomposite after systemic injection, mice (Charles River Laboratories)were intravenously treated with 200 μCi of the unlabeled DTC/RLuc orDTC-Apo/RLuc, or naked phRL-CMV or a 5% dextrose solution, as controls,and sacrificed the following day. Heart, lung, kidney and liver wereeach harvested from each mouse and homogenized. The bioluminescentintensity of each tissue homogenate was measured using a renillaluciferase assay system (Promega) to determine the luciferase expressionlevel per total protein. The results are shown in FIG. 2C.

As shown in FIG. 2C, consistently with liver-specific accumulation ofisotope or rhodamine-labeled DTC-Apo composites (FIGS. 2A and 2B),luminescence signals are particularly prominent in the liver of miceinjected with DTC-Apo/RLuc in an amount ranging from 6,700 to 50,300RLU/mg. In contrast, in mice treated with DTC/RLuc, luciferase signalswere strong in the lung and kidney but only modest in the liver.

The results suggest that the inventive composite can liver-specificallydeliver a therapeutic gene or drug to hepatic cells the therapeutic genebeing expressed therein.

TEST EXAMPLE 4 Therapeutic Effect of the Inventive Composite withNucleic Acid

To examine the therapeutic activity of the inventive composite, in vivoantiviral effect of DTC-Apo containing HBV-specific siRNA(DTC-Apo/siHBV) was examined using a mouse model for acute HBV infectionas follows.

First, in order to establish an acute HBV-infected mouse model, HBVreplication competent plasmid, pCpGHBV-MBRI, was created by excision ofthe viral genome from the mother clone pHBV-MBRI (Shin, D., et al.,Virus Res. (2006), 119: 146-153) and religated into SpeI and XbaIdigested pCpG-mcs (InvivoGen, USA), which is known to be not induciblenonspecific inflammatory responses in mammalian hosts. Then, 10 μg ofpCpGHBV-MBRI was hydrodynamically injected into the tail veins of femaleC57BL/6 mice (Charles River Laboratories) of 8-9 weeks of age weighingapproximately 20 g to induce the acute HBV infectious. After 8 hours,the HBV-infected model mice were intravenously administered with 2 mg/kg(i. e., 40 μg of nucleic acid per mouse) of DTC-Apo/siHBV,DTC-Apo/siCont, DTC/siHBV and DTC/siCont obtained in Examples 2 and 5,and Comparative Examples 1 and 4, respectively. 2 mg/kg of nakedHBV-specific siRNA or 5% dextrose solution was also administrated tocontrol mice groups. Serum samples were collected from each treatedmouse on days 2, 4, 6 and 8 after injection, and the serum HBV surfaceantigen (HBsAg) level, one of the major viral structural proteins, wasdetermined by ELISA (DiaSorin, USA) to monitor the viral protein levelsecreted into the blood. The results are shown in FIG. 3A.

As shown in FIG. 3A, there is a significant reduction of serum HBsAg inmice administered with a single dose of DTC-Apo/siHBV particles, asshown by the average inhibitions degree of 65.1% (P=0.014), 63.4%(P=0.047), 74.9% (P=0.015) and 72.8% (P=0.034) on days 2, 4, 6 and 8post-injection, respectively, relative to the matched DTC/siHBV orDTC-Apo/siCont treated mice.

Further, in order to examine the dose-dependent activity ofDTC-Apo/siHBV, the mice with acute HBV replication were treated with0.5, 1, or 2 mg/kg doses of DTC-Apo/siHBV, while 2 mg/kg ofDTC-Apo/siCont or a 5% dextrose solution was also administrated to themodel mice as control groups. The serum viral antigen levels in eachmouse was monitored at day 4 post-injection. The results are shown inFIG. 3B.

As shown in FIG. 3B, the treatment of the inventive composite containingHBV-specific siRNA reduces the viral antigen expression in mice withacute HBV replication in a does-dependent manner, unlike the controlgroups.

These in vivo data indicate that the inventive composite can promote thehepatic tissue-specific delivery of a therapeutic nucleic acid or drugand lead to potent therapeutic effects in vivo, only through aintravenous treatment of the inventive composite containing atherapeutic nucleic acid or drug.

TEST EXAMPLE 5 Therapeutic Effect of the Inventive Composite withNucleic Acid

In order to confirm that the target-specific effect of the inventivecomposite comprising a therapeutic nucleic acid or drug occursselectively and mainly in the hepatic tissue, 6-7-week-old female Balb/cmice (Charles River Laboratories) were hydrodynamically injected with 10μg of pEGFPLuc plasmid (Clontech), which is known to express the fireflyluciferase and also to facilitate in vivo image analysis, respectively.

After one day, DTC-Apo/siLuc or DTC-Apo/siCont obtained in Example 3 or5, or a 5% dextrose solution control was injected at a dose of 1 mg/kgvia the tail veins of the mice under ambient pressure, and the followingday, the treated mice were anaesthetized with 2% isoflurane, andintraperitoneally injected with 200 μl of 15 mg/ml D-luciferin(Molecular Imaging Products Company, USA). Ten minutes later, photonsignals from the whole body of each mouse was analyzed using an IVISimaging system (Xenogen). The results are shown in FIGS. 4A and 4B.

As shown in FIGS. 4A and 4B, there is no signal change suggestingluciferase expression inhibition in mice injected with DTC-Apo/siCont,while a dramatic reduction in luciferase activity (about 70%) wasobserved for mice treated with DTC-Apo/siLuc as early as day 1 aftertreatment.

Taken together, these results show that the selective target of theinventive composite administered systemically is the liver and that thetherapeutic nucleic acid or drug delivered by the inventive composite isspecifically released into hepatic cells to manifest a potenttherapeutic effect, without inducing any enzymatic abnormalities orpathological damage of the normal liver function.

While the embodiments of the subject invention have been described andillustrated, it is obvious that various changes and modifications can bemade therein without departing from the spirit of the present inventionwhich should be limited only by the scope of the appended claims.

1. A composite comprising an apolipoprotein A-I and a liposome-formingmaterial.
 2. The composite of claim 1, wherein the liposome-formingmaterial is a cationic or neutral liposome-forming material, or amixture of thereof.
 3. The composite of claim 2, wherein the cationicliposome-forming material is selected from the group consisting of DOTAP(1,2-dioleoyl-3-trimethylammonium-propane), DC-cholosterol(3β-[N-(N′,N′-dimethylaminoethane)carbamyl]cholesterol), DDAB(dimethyldioctadecylammonium bromide), and a mixture thereof.
 4. Thecomposite of claim 2, wherein the neutral liposome-forming material isselected from the group consisting of DOPE (L-alpha-dioleoylphosphatidylethanolamine), cholesterol, and a mixture thereof.
 5. Thecomposite of claim 1, wherein the weight ratio of the apolipoprotein A-Iand the liposome-forming material is in the range of 1:0.1 to 1:1000. 6.The composite of claim 1, which further comprise a therapeutic nucleicacid or a drug.
 7. The composite of claim 6, wherein the therapeuticnucleic acid is selected from the group consisting of a DNA, an RNA, anda derivative thereof.
 8. The composite of claim 7, wherein the RNA is ansiRNA specific for HBV or HCV genome.
 9. The composite of claim 6,wherein the therapeutic drug is an active polypeptide, an anticanceragent or an antivirus agent.
 10. The composite of claim 9, wherein theactive polypeptide is selected from the group consisting of epidermalgrowth factor (EGF), erythropoietin (EPO), coagulation factors VIII, IXand VIIa, follicle stimulating hormone (FSH), granulocytecolony-stimulating factor (GCSF), granulocyte-macrophage colonystimulating factor (GM-CSF), insulin, insulin-like growth factor (IGF),interferon-α, -β and -γ (IFN-α, -β and -γ), interleukin-1, -2, -11, -12and -15 (IL-1, -2, -11, -12 and -15), parathyroid hormone (PTH),platelet-derived growth factor (PDGF), human growth hormone (hGH),tissue plasminogen activator (tPA), vascular endothelial growth factor(VEGF), and a mixture thereof.
 11. The composite of claim 9, wherein theanticancer agent is selected from the group consisting of carboplatin,cisplatin, oxaliplatin, heptaplatin, etoposide, semustine,hydroxycarbamide, citarabine, fludarabine, doxorubicin, epirubicin,idarubicin, pirarubicin, fluorouracil (5-FU), fluoxuridine, mitomycin,bleomycin, clofazimine, interferon, streptozocin, gemcitabine,enocitabine, capecitabine, ursodeoxycholic acid, sorafenib, tegafur,holmium, a holmium-chitosan complex, and a mixture thereof.
 12. Thecomposite of claim 9, wherein the antivirus agent is selected from thegroup consisting of atazanavir, ribavirin, zanamivir, acyclovir,entecavir, didanosin, nevirapine, valaciclovir, nelfinavir, efavirenz,ganciclovir, lamivudine, famciclovir, stavudine, abacavir, indinavir,oseltamivir, inosiplex, adefovir, and a mixture thereof.
 13. Acomposition comprising the composite of claim 1 and a pharmaceuticallyacceptable carrier.
 14. The composition of claim 13, which furthercomprise a therapeutic nucleic acid or a drug.
 15. The composition ofclaim 14, wherein the therapeutic nucleic acid is selected from thegroup consisting of a DNA, an RNA, and a derivative thereof.
 16. Thecomposition of claim 15, wherein the RNA is an siRNA specific for HBV orHCV genome.
 17. The composition of claim 14, wherein the therapeuticdrug is an active polypeptide, an anticancer agent or an antivirusagent.
 18. The composition of claim 17, wherein the active polypeptideis selected from the group consisting of epidermal growth factor (EGF),erythropoietin (EPO), coagulation factors VIII, IX and VIIa, folliclestimulating hormone (FSH), granulocyte colony-stimulating factor (GCSF),granulocyte-macrophage colony stimulating factor (GM-CSF), insulin,insulin-like growth factor (IGF), interferon-α, -β and -γ (IFN-α, -β and-γ), interleukin-1, -2, -11, -12 and -15 (IL-1, -2, -11, -12 and -15),parathyroid hormone (PTH), platelet-derived growth factor (PDGF), humangrowth hormone (hGH), tissue plasminogen activator (tPA), vascularendothelial growth factor (VEGF), and a mixture thereof.
 19. Thecomposition of claim 17, wherein the anticancer agent is selected fromthe group consisting of carboplatin, cisplatin, oxaliplatin,heptaplatin, etoposide, semustine, hydroxycarbamide, citarabine,fludarabine, doxorubicin, epirubicin, idarubicin, pirarubicin,fluorouracil (5-FU), fluoxuridine, mitomycin, bleomycin, clofazimine,interferon, streptozocin, gemcitabine, enocitabine, capecitabine,ursodeoxycholic acid, sorafenib, tegafur, holmium, a holmium-chitosancomplex, and a mixture thereof.
 20. The composition of claim 17, whereinthe antivirus agent is selected from the group consisting of atazanavir,ribavirin, zanamivir, acyclovir, entecavir, didanosin, nevirapine,valaciclovir, nelfinavir, efavirenz, ganciclovir, lamivudine,famciclovir, stavudine, abacavir, indinavir, oseltamivir, inosiplex,adefovir, and a mixture thereof.